DNA-directed RNA Polymerase II (RNAPII) is highly conserved among eukaryotic organisms and plays a fundamental role in cellular life, specifically gene transcription. During transcription, RNAPII transcribes genes into messenger RNA, a process aided by the general transcription factors: TBP, TFIIA, TFIIE, TFIIH, TFIIB and TFIIF. Meaningful biochemical experiments have demonstrated that transcription initiation is a highly dynamic event, with the possibility of discrete stages where the general transcription factors recognize, melt and load a nucleic acid scaffold (NAS) inside RNAPII's active site. This process is universal, for all eukaryotic species, and is at the core of gene regulation; therefore, understanding its molecular details will provide essential clues that could potentially lead to pharmacological manipulation of gene expression. Now that present technology enables us to crystallize and solve high-resolution structures of large multicomponent complexes, we can begin to examine the transcriptional machinery at the atomic level and address this specific question: What are the roles of the general transcription factors, TFIIB and TFIIF, in DNA stabilization during transcription bubble loading and promoter escape? The intention of this proposal is to answer this question using novel methodologies in chemical cross-linking, X-ray crystallography, and NMR. In a two prone approach we will first build on preliminary data which has shown that it is possible to reconstitute and crystallize a stoichiometric RNAPII - TFIIF complex, as well as obtain anisotropic diffraction to 2.9 . To improve crystallization conditions we will implore a novel chemical-crosslinking methodology, which will help prevent complex dissociation during crystallography trials. Secondly, we will demonstrate the ability to reconstitute early transcribing intermediates comprised of TFIIB, TFIIF, the NAS, and RNAPII, and investigate how the individual factors affect the opening/closing ends of the NAS using fluorescent probes. We will also characterize the interactions of the TFIIF ?-subunit with the NAS and TFIIB using NMR, which will provide structural insight into why certain mutants within this subunit promote transcription defects. Our strong preliminary results, distinctive understanding of the complete nucleic acid scaffold, and track record working with multi-protein complexes, places us in a unique position to pursue this line of research. Once accomplished, these goals will enhance our understanding of the intricate mechanisms by which general transcription factors cooperatively interact with RNAPII to initiate transcription, and provide a blueprint for studying novel multi-protein complexes implicated in disease.